The long-term objectives of this project are to understand the signaling mechanisms of the Dbl family guanine nucleotide exchange factors (GEFs) and to examine the involvement of the GEF abnormalities in human diseases. The Dbl related GEFs represent a large family of cell growth regulatory molecules with over 70 mammalian members. Their cellular functions intimately depend on their ability to interact and activate specific Rho GTPases, leading to various physiological responses including cytokinesis, cell movement, cell proliferation and survival. The Dbl family members share the structural module of a Dbl-homology (DH) domain in tandem at the carboxyl terminus with a Pleckstrin-homology (PH) domain. Previous studies have established that the DH domain is responsible for Rho GTPase recognition and the GEF activity, whereas the PH domain, together with additional regulatory motifs, is involved in intracellular targeting and/or modulation of the DH domain function. To continue the lines of studies of our last funding period, we plan to pursue three specific aims to examine whether oncogenic activation of the GEFs might be associated with cancer development and to utilize the mechanistic features to devise ways to interfere with the Dbl family GEF signaling. In the first aim we will examine the signaling mechanisms of two members of the Dbl family, Common Site lymphoma/leukemia GEF (Clg) and Leukemia associated Rho GEF (LARG), in the physiologically and pathologically relevant primary hematopoietic stem/progenitor cells. In the second aim we will seek a functional association of Clg and the fusion MLL-LARG activation with leukemogenesis in a mouse transplant model. In the third aim we will devise peptide and small molecule inhibitors of the GEF-Rho GTPase interaction based on the structure-function knowledge. Together, these mechanistic and functional studies will provide valuable information on the physiological/pathological roles of Dbl family GEFs and draw a close connection between the Rho GTPase activators and human pathological conditions such as cancer. The results may also provide valuable pharmacological reagents for the intervention of the GEF-mediated signaling.